Weber, MichaelMichaelWebervon der Emde, HenrikHenrikvon der EmdeLeutenegger, MarcelMarcelLeuteneggerGunkel, PhilipPhilipGunkelSambandan, SivakumarSivakumarSambandanKhan, Taukeer A.Taukeer A.KhanKeller-Findeisen, JanJanKeller-FindeisenCordes, Volker C.Volker C.CordesHell, Stefan W.Stefan W.Hell2022-12-012022-12-012022https://resolver.sub.uni-goettingen.de/purl?gro-2/118008Abstract Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power. By blue-shifting the STED beam and separating fluorophores by on/off switching, individual fluorophores bound to a DNA strand are localized with σ  = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Because our experiments yield a localization precision σ  = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up new areas of application in the study of macromolecular complexes in cells.enhttps://creativecommons.org/licenses/by/4.0journal_article10.1038/s41587-022-01519-41519